Molecular characterisation of fowl adenovirus associated with hydropericardium hepatitis syndrome in broiler and layer breeders in Azerbaijan.

BACKGROUND: Fowl adenovirus-4 is a causative agent of hydropericardium hepatitis syndrome (HHS) in chickens and has been frequently reported from many countries. Fowl adenoviruses cause severe disease and mortality in broiler and layer breeders in Azerbaijan. Therefore, in this study, pathological lesions and the dissemination of fowl adenovirus-4 into the visceral organs of infected birds were investigated as well as molecular characterisation of detected strains. For this, liver, heart and spleen from 20 necropsied chickens originated from a broiler breeder flock and a layer breeder flock were embeded on the FTA cards and the samples were analysed for adenovirus-DNA by PCR and sequencing. RESULTS: The findings of necropsy in both broiler and layer breeder chickens were similar, and the liver was severely effected showing hepatitis, and the heart with hydropericardium lesions. The kidneys were swollen with haemorrhages and small white foci on the surface of the spleens were noted. Intestinal congestion and ecchymotic hemorrhages were also observed in some birds. Fowl adenovirus-4-DNA was detected by PCR in all collected organs of 20 birds. The sequence analysis revealed that fowl adenovirus-4 present in Azerbaijan and close similarity of the hexon genes of the adenoviruses existing in the Middle East, North America, far east and Indian subcontinent were determined by phylogenetic analysis. However, sequence diversity was detected from the adenovirus strains circulating in Europe, North and South America. CONCLUSIONS: This study indicates the impact of fowl adenovirus-4 on the poultry health and production, and improved disease control and prevention strategies are necessary to reduce the HHS disease in chickens in Azerbaijan.

Chicken Interferon-Alpha and -Lambda Exhibit Antiviral Effects against Fowl Adenovirus Serotype 4 in Leghorn Male Hepatocellular Cells.

Hydropericardium hepatitis syndrome (HHS) is primarily caused by fowl adenovirus serotype 4 (FAdV-4), causing high mortality in chickens. Although vaccination strategies against FAdV-4 have been adopted, HHS still occurs sporadically. Furthermore, no effective drugs are available for controlling FAdV-4 infection. However, type I and III interferon (IFN) are crucial therapeutic agents against viral infection. The following experiments were conducted to investigate the inhibitory effect of chicken IFN against FadV-4. We expressed recombinant chicken type I IFN-α (ChIFN-α) and type III IFN-λ (ChIFN-λ) in Escherichia coli and systemically investigated their antiviral activity against FAdV-4 infection in Leghorn male hepatocellular (LMH) cells. ChIFN-α and ChIFN-λ dose dependently inhibited FAdV-4 replication in LMH cells. Compared with ChIFN-λ, ChIFN-α more significantly inhibited viral genome transcription but less significantly suppressed FAdV-4 release. ChIFN-α- and ChIFN-λ-induced IFN-stimulated gene (ISG) expression, such as PKR, ZAP, IRF7, MX1, Viperin, IFIT5, OASL, and IFI6, in LMH cells; however, ChIFN-α induced a stronger expression level than ChIFN-λ. Thus, our data revealed that ChIFN-α and ChIFN-λ might trigger different ISG expression levels, inhibiting FAdV-4 replication via different steps of the FAdV-4 lifecycle, which furthers the potential applications of IFN antiviral drugs in chickens.

Transcriptome analysis of fowl adenovirus serotype 4 infection in chickens.

Fowl adenovirus serotype 4 (FAdV-4) is a causative agent of inclusion body hepatitis and hydropericardium-hepatitis syndrome. These diseases cause considerable economic losses in the global poultry industry and are significant stressors for infected chickens. However, the molecular mechanisms of FAdV-4 pathogenesis are poorly understood. In the present study, we identified differentially expressed genes from the livers of FAdV-4-infected chickens using RNA-seq at 7, 14 and 21 days after FAdV-4 infection. We identified 2395 differentially expressed genes at the three time points. These genes were enriched in variety of biological processes and pathways including PPAR and Notch signaling, cytokine-cytokine receptor interactions and Toll-like receptor signaling pathways. The transcriptional data were validated by quantitative real-time PCR. Our results will assist in the understanding of the molecular pathogenesis of FAdV-4 infection and for developing novel antiviral therapies.

The complete genome sequence of a new fowl adenovirus (Fadv-4) strain from a recent outbreak in a chicken farm in Iran.

The whole genomic sequence of fowl adenovirus C (FAdV-4) strain FAdV-4/Pasouk, isolated from chickens with hepatitis-hydropericardium syndrome (HHS) from an outbreak in Iran, has been deposited in GenBank under accession number ON652872. Notably, this FAdV-4 isolate exhibited significant genetic similarities to contemporary isolates originating from China, indicating a shared ancestry.

Genetic evolution of fowl adenovirus serotype 4 and its pathogenicity to Cherry Valley ducks in China.

Hydropericardium hepatitis syndrome (HHS), a novel poultry disease, is caused by fowl adenovirus 4 (FAdV-4). It mainly infects 3-5-week-old broilers. In July 2015, the first outbreak of HHS occurred in the broilers in east China, which caused great economic losses to the poultry industry. In June 2019, infectious disease was detected with suspected HHS symptoms on a duck farm in Linyi City, Shandong Province. The main necropsy lesions included pericardial effusion and hepatitis. In this study, we isolated a strain of FAdV-4 from naturally infected ducks and named it SDLY190604, and the hexon gene sequence was amplified and analyzed using polymerase chain reaction (PCR). In order to study the effect of FAdV-4 on Cherry Valley ducks, we inoculated three-week-old ducks with 0.2 ml of FAdV-4 virus fluid (TCID50 of 10-6.3/0.1 ml) by orally, subcutaneously and intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines and viral load were detected and recorded within 15 days after infection. The results showed that ducks in the experimental groups exhibited typical symptoms of hydropericardium and hepatitis. The histopathological sections showed multiple-organ damage, including serious liver and kidney damage with elevated levels of inflammatory cytokines, probably due to the infection and innate immune response. Later, immunosuppression occurred, resulting in decreased levels of cytokines. The viral load indicated that the virus could be present in several organs of the ducks, with the highest viral DNA found in the liver, followed by the kidney. Compared to the subcutaneous and oral groups, the intramuscular group exhibited the highest viral load. In summary, this study can increase our understanding of the pathogenicity of FAdV-4 in ducks and provide a basis for further understanding of the virus, imparting new insights into disease research.

Molecular characterization and pathogenicity of a fowl adenovirus serotype 4 isolated from peacocks associated with hydropericardium hepatitis syndrome.

In September 2019, a highly prevalent infectious disease caused severe hydropericardium hepatitis syndrome (HHS) in a peacock farm in Central China. The disease showed high mortality of 78.6% in 28-42 day-old peacocks. In this study, one strain of highly pathogenic fowl adenovirus serotype 4 (FAdV-4) was isolated from peacocks and designated as HN19. Molecular characterization of amino acid revealed that HN19 contains the same deletions as the dominate strains in chickens in China recently. Phylogenetic analyses revealed that HN19 showed higher homology with other FAdV-4 strains isolated from China, indicating that HN19 might originate from previously FAdV-4 predecessor in China. Experimental infection of the HN19 strain via intramuscular injection led to 100% mortality rate in 21-day-old specific pathogenic-free (SPF) chickens. To our knowledge, this represents the first report on the prevalence of FAdV-4 in peacocks. These results suggested that the potential risk of cross-species transmission of FAdV-4 from chickens to peacocks, highlighting the need for implementing strict biosecurity measures to avoid the mixing of different bird species.

Concurrent infection of Avibacterium paragallinarum and fowl adenovirus in layer chickens.

The diagnosis of a concurrent infection of Avibacterium paragallinarum and fowl adenovirus (FAdV) in an infectious coryza-like outbreak in the outskirt of Beijing is reported. The primary signs of the infection were acute respiratory signs, a drop in egg production, and the presence of hydropericardium-hepatitis syndrome-like gross lesions. Laboratory examination confirmed the presence of A. paragallinarum by bacterial isolation and a species-specific PCR test. In addition, conventional serotyping identified the isolates as Page serovar A. Fowl adenovirus was isolated from chicken liver specimen and identified by hexon gene amplification. In addition, histopathologic analysis and transmission electron microscopy examination further confirmed the presence of the virus. Both hexon gene sequencing and phylogenetic analysis defined the viral isolate as FAdV-4. The pathogenic role of A. paragallinarum and FAdV was evaluated by experimental infection of specific-pathogen-free chickens. The challenge trial showed that combined A. paragallinarum and FAdV infection resulted in more severe clinical signs than that by FAdV infection alone. The concurrent infection caused 50% mortality compared with 40% mortality by FAdV infection alone and zero mortality by A. paragallinarum infection alone. To our knowledge, this is the first report of A. paragallinarum coinfection with FAdV. The case implies that concurrent infections with these 2 agents do occur and more attention should be given to the potential of multiple agents during disease diagnosis and treatment.

Pathogenicity of a fowl adenovirus serotype 4 isolated from chickens associated with hydropericardium-hepatitis syndrome in China.

Hydropericardium-hepatitis syndrome (HHS) is characterized by pericardial effusion and hepatitis and causes huge economic losses in the poultry industry in China. In this study, a strain of fowl adenoviruses (FAdV-4) (GX-1) was isolated from liver samples of diseased chickens with HHS. Phylogenetic analysis based on complete genome gene revealed that GX-1 clustered with the C-type fowl adenovirus and was serotyped as FAdV-4. Pathogenicity testing showed that the GX-1 strain caused 100% mortality in 10-day-old specific pathogen-free chickens at a dose of 104 tissue culture infective doses (TCID50) within 3 d post-infection. A viral dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were flabby with amber-colored and jelly-like fluid in the pericardial sacs. The kidneys were swollen and congested. Histologically eosinophilic intranuclear inclusion body could be seen in the hepatic cell. The result of histopathological examination also revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging. Other tissues like kidney, bursa of Fabricius, thymus, and spleen were observed degeneration and necrosis. Virus-specific antibodies appeared in serum beginning at day 14 and reached statistically significant levels at 21, 28, 35, and 42 dpi (P < 0.001). In conclusion, we identified a highly virulent FAdV-4 virus as causative agent of the HHS outbreak reported here. The FAdV-4 GX-1 strain will be valuable for vaccine evaluation and development to prevent and reduce the spread of HHS in the poultry industry.

Identification, Pathogenicity of Novel Fowl Adenovirus Serotype 4 SDJN0105 in Shandong, China and Immunoprotective Evaluation of the Newly Developed Inactivated Oil-emulsion FAdV-4 Vaccine.

Since mid-2015, numerous outbreaks of hydropericardium-hepatitis syndrome (HHS), which is caused by a novel fowl adenovirus serotype 4 (FAdV-4), have been reported in chickens in parts of China, thereby causing huge economic losses to the poultry industry. Thus, an effective vaccine to control the further spread of infections with this hyper-virulent FAdV-4 is imperative. In this study, we isolated a novel FAdV-4 strain SDJN0105 from a broiler farm with HHS disease in Shandong Province. Pathogenicity was evaluated by the observation of clinical symptoms, necropsy changes, and pathological tissue sections after oral and intramuscular (IM) infection of Specific pathogen free (SPF) chickens. The chickens infected by IM injection all died within three days, and chickens infected via the oculonasal route died within five days post-infection (dpi). Histopathological examination revealed that the pathology was confined to heart, liver, spleen, lung, kidney, and particularly the liver. Irrespective of the inoculation route, the highest viral DNA copy numbers were detected in the livers of infected chickens. The mRNA expression levels of IL-1ß, IL-6, IL-8, IFNs, TNF-α, Mx, and OASL were significantly upregulated during the viral infection. In addition, an inactivated oil-emulsion FAdV-4 vaccine was developed. The vaccine could provide full protection for SPF chickens against a lethal dose of the FAdV-4 strain SDJN0105 and a high level of antibodies. These results improve our understanding of the innate immune responses in chickens infected with FAdV-4 and the pathogenesis of FAdV-4 caused by host factors, and the developed FAdV-4 vaccine is promising as a drug candidate for the prevention and reduction of the spread of HHS in poultry in China.

Prevalence of Fowl Adenovirus Serotype 4 and Co-Infection by Immunosuppressive Viruses in Fowl with Hydropericardium Hepatitis Syndrome in Shandong Province, China.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.

Characterization of fowl adenovirus serotype 4 circulating in chickens in China.

Outbreaks of fowl adenovirus (FAdV) has resulted in huge economic losses in poultry industry in China since 2015. This study detected the pathogens from diseased chickens and determined that fowl adenovirus serotype 4 (FAdV-4) and co-infection of immunosuppressive pathogens were the causes of the outbreaks. Phylogenetic analysis results indicated that these pandemic strains originated from previously FAdV-4 predecessor in China and had obtain gene mutations that might contribute to enhanced pathogenicity of these strains. Compared with early strains, the pathogenicity of novel FAdV-4 strains significantly increased, which led to systemic infections and injuries to multiple organs in the infected chickens. Our study could provide useful information for understanding of the FAdV-4 and favorable theory basis for clinical prevention and control of the disease.

Pathogenic, Phylogenetic, and Serological Analysis of Group I Fowl Adenovirus Serotype 4 SDSX Isolated From Shandong, China.

Hydropericardium hepatitis syndrome (HHS) caused by group I fowl adenovirus serotype 4 (FAdV-4) is an acute and infectious disease in fowl, particularly in broilers aged 3-5 weeks. In June 2015, a highly pathogenic disease outbroke in 25-40 day-old ducklings in Shandong province, characterized similar symptom to HHS. In order to determine the pathogenic mechanism of FAdV-4 (SDSX strain) in meat ducks. We divided 90 25-day-old Cherry Valley meat ducks into three groups (oral, subcutaneous, and control; 30 ducks in each group) and infected them with the virus. HHS, inclusion body hepatitis, and enlargement and hemorrhage of the spleen, kidney, lung, thymus, and brain were observed in FAdV-4-infected meat ducks. Histopathological changes were mainly characterized by severe fatty degeneration in the liver, basophilic inclusion bodies in hepatocytes, and vacuolation in the bursa. More importantly, viral DNA could be detected by quantitative real-time polymerase chain reaction in several viscera tissues (e.g., heart, liver, spleen) on the third day after infection. Notably, the livers of the two infected groups contained the highest concentration of viral DNA. In addition, immune responses were studied based on titer levels of the virus antibody and the levels of inflammatory cytokines interleukin (IL)-2 and interferon (IFN)-γ, and most levels were significantly upregulated, indicating that the host immune responses were activated early in infection. These findings increase our understanding of the pathogenicity of FAdV-4 (SDSX) in meat ducks and provide the foundation for further in-depth study of the pathogenic mechanism of this virus.

Fowl Adenovirus Serotype 4 SD0828 Infections Causes High Mortality Rate and Cytokine Levels in Specific Pathogen-Free Chickens Compared to Ducks.

Hydropericardium syndrome and inclusion body hepatitis, together called hydropericardium-hepatitis syndrome, are acute infectious diseases found in chickens. These diseases are caused primarily by fowl adenovirus serotype 4 (FAdV-4) strains. In this study, we isolated a FAdV-4 strain (SD0828) from clinically diseased chickens and phylogenetically analyzed the L1 loops of the hexon protein sequences in 3-week-old specific pathogen-free chickens and ducks infected intramuscularly and orally, determining differences in the pathogenicity by observing clinical signs and gross and histological lesions. We also detected the viral load in tissue samples. Postinfection necropsy showed that all chickens but no ducks exhibited typical necropsy lesions. Additionally, all chickens infected intramuscularly died within 2 days postinfection (dpi), and all those infected orally died within 5 dpi, whereas no infected ducks died before 28 dpi. Quantitative real-time polymerase chain reaction analysis was used to determine the viral load in the tissues of hearts, livers, spleens, lungs, and kidneys and in cloacal cotton swabs from infected chickens and ducks at 1, 2, 3, 5, 7, 14, 21, and 28 dpi. The greatest number of viral DNA copies was found in the livers of infected chickens, yet no virus was found in any samples from infected ducks. In addition, the viral load increased over time in both chicken and duck embryo fibroblasts (CEFs and DEFs, respectively); in the former, replication speed was significantly greater than in the latter. Innate immune responses were also studied, both in vivo and in vitro. In CEFs, DEFs, and chickens infected intramuscularly, but not in infected ducks, mRNA expression levels of proinflammatory cytokines (interleukin-6 and -8) and interferon-stimulated genes (Mx and OAS) were significantly upregulated. Although some cytokines showed significant upregulation in the oral chickens, most did not change significantly. Finally, the duck retinoic acid-inducible gene I and its caspase activation and recruitment domain both had significant antiviral functions in CEFs, particularly after 24 h postinfection. Taken together, this research provides new insights into the interactions between FAdV-4 and the innate immune systems of studied hosts (chickens and ducks).

Hydropericardium Hepatitis Syndrome Emerged in Cherry Valley Ducks in China.

Since June 2015, a highly pathogenic disease occurred in duck flocks in China, causing pericardial effusion, enlarged discoloured liver, renal enlargement and haemorrhagic lung with a mortality ranging from 5% to 20%. Previous study confirmed that Fowl adenovirus group C (FAdV-C) and some field FAdVs isolates had been identified as causative agents of hydropericardium hepatitis syndrome (HHS) in chickens and geese world widely. In this study, we firstly report the isolation of FAdV-C from ducks with HHS. The two isolates, designated as SDSX and SDJX, were separated from liver samples using 9-day-old SPF chicken embryos and could cause severe cytopathic effects in duck and chicken embryonic kidney cells. The entire ORF sequences of hexon gene of the two isolates were amplified, sequenced and analysed by restriction fragment length polymorphism. Phylogenetic analysis of loop 1 sequences of hexon gene of FAdVs revealed that the two isolates were closely related to FAdV-C isolates, which could cause HHS in chickens. Experimental infection indicated that the isolate was high pathogenicity to 20-day-old ducks. Our study shows that the recently emerged HHS in ducks was caused by FAdV-C and may possess a potential risk to other poultry flocks.

Fiber gene based molecular and biological characterization of hydropericardium-hepatitis syndrome associated avian adenoviruses.

This study was designed to perform biological and molecular characterization of avian adenoviruses (AAVs) recovered from suspected cases of hydropericardium-hepatitis syndrome (HHS) in commercial poultry. Initially the samples were screened by Agar Gel Precipitation Test (AGPT) for the presence of AAVs followed by its confirmation and typing through polymerase chain reaction (PCR) focusing on already reported serotypes AAV-4, AAV-8 and AAV-10 elsewhere. These PCR-positive samples were further subjected to amplification of fiber gene, followed by conducting restriction fragment length polymorphism (RFLP) using restriction enzyme Alu. The selected isolates were further propagated through cell culture and pathogenic potential of selected isolates was determined by infecting chickens. In this study, out of a total 190 samples, 57.8% of suspected cases were found positive for AAV presence through AGPT while sub-type identification using PCR revealed 46.3% for these viruses belonging to AAV-4, 41.8% to AAV-8 and 11.8% showed co-infection of AAV-4 and AAV-8. AAV-10 was not detected in any of the tested samples. On the basis of RFLP pattern, AAV-4 isolates were further divided into four sub-groups (A-D) while AAV-8 isolates had identical RFLP pattern. To further evaluate the pathogenic potential of these sub-groups of AAV-4 isolates, specific pathogen free (SPF) chicks were challenged with selected isolates belonging to each of the sub-groups, resulting in variable pattern of pathogenicity. It is concluded that any variation in the fiber gene of AAV-4 isolates may affect its pathogenicity and eventually specificity of the vaccines used against such infections. Therefore, regular monitoring of the circulating AAV serotypes may be helpful in understanding the pathogenic potential of emerging AAVs, which may lead to development of more effective response strategies accordingly.

Cell-culture derived fowl adenovirus serotype 4 inactivated vaccine provides complete protection for virus infection on SPF chickens.

Inclusion body hepatitis and hepatitis-hydropericardium syndrome caused by high-pathogenic fowl adenovirus serotype 4 has recently plagued Chinese poultry industry and caused huge economic losses since 2013. So far, there is no commercial vaccine available to control this disease. In this study, we reported the development of both embryo-adapted and cell-culture derived inactivated FAdV-4 vaccines and evaluated their efficacies in chicken. Compared to embryo-adapted vaccine, cell-culture derived vaccine induced significantly earlier and higher serological response measured by AGP and ELISA. After virus challenge, chicken immunized with cell-culture derived vaccine did not showed any gross and histopathological lesions, whereas inclusion body hepatitis was observed in the liver of chicken vaccinated with embryo-adapted vaccine. No mortality was observed in both the vaccinated groups. The above results suggested that cell-culture derived FAdV-4 inactivated vaccine could be a better vaccine candidate than embryo-adapted vaccine to control FADV-4 infections in China.